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Permanent monitoring of pyrrolizidine alkaloid content in micropropagated Tussilago farfara L.: A tool to fulfill statutory demands for the quality of coltsfoot in Austria and Germany

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Abstract

For several years the use of the medicinal plant Tussilago farfara (coltsfoot) has been restricted in Austria and Germany due to the content of toxic pyrrolizidine alkaloids. In order to meet the demand for crude drug an in vitro collection of clones was established as a basis for the selection of suitable lines. Analyses of the alkaloid content showed that some clones were free of the toxic compounds. One of these clones has now been cultivated in the field for several years, and permanent monitoring confirms that the quality is in accordance with the requirements of the legislation. By large scale culture of the variety Tussilago farfara 'Wien' it is now possible to provide material for pharmaceutical and dietary purposes and thus to re-introduce this medicinal plant to the market.

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... According to the literature, the content of toxic PAs in coltsfoot leaves is very changeable and ranges from 0.1 to 368 μg g −121222326,27]. It seems that such a large variation of the amount of PAs can arise from the different origin of plant material (commercial samples or samples from natural sites) and its preparation (raw material drying method) as well as from different extraction methods (with water or methanol, citric acid, ammonia, etc.) and analytical methods (capillary zone electrophoresis, gas or liquid chromatography). However, phytochemical analysis of plant material from various provenances in Europe shows the high variability in the content of PAs in coltsfoot [22,27]. ...
... It seems that such a large variation of the amount of PAs can arise from the different origin of plant material (commercial samples or samples from natural sites) and its preparation (raw material drying method) as well as from different extraction methods (with water or methanol, citric acid, ammonia, etc.) and analytical methods (capillary zone electrophoresis, gas or liquid chromatography). However, phytochemical analysis of plant material from various provenances in Europe shows the high variability in the content of PAs in coltsfoot [22,27]. Our study of samples from natural populations in Poland confirms these results (Tab. ...
... Lebada et al. [22]). However, a PA-free clone of coltsfoot, registered as the variety T. farfara 'Wien' , is known in Austria and Germany [27]. In the context of the use of coltsfoot leaves as food, the problem of the thermal stability of PAs is interesting. ...
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Coltsfoot (Tussilago farfara L.) is a common species, widely used in European and Chinese traditional medicine for the treatment of respiratory diseases. However, raw material from this plant contains hepatotoxic pyrrolizidine alkaloids (PAs). The aim of the study was to determine the variability of the level of PAs (senkirkine and senecionine) in leaves of coltsfoot originated from natural populations in Poland. In the phytochemical analysis, 20 samples of T. farfara were used. This plant material was obtained from the Garden of Medicinal Plants in Plewiska near Poznań and originated from different regions of Poland. Coltsfoot leaves were harvested in the middle of July of 2010 and then dried at room temperature. The alkaloid content was detected using the HPLC-DAD method. The amount of PAs in leaves of T. farfara changed in a wide range from 0.06 to 1.04 μg g−1 of dry matter (DM). The content of senkirkine and senecionine was positively correlated (r = 0.68, P = 0.001). There was no statistically significant correlation between the amount of PAs as well as leaf weight and water content in leaves of T. farfara. Our results showed that a medium-sized leaf of coltsfoot (0.33 g DM) may contain from 0.02 to 0.34 μg of PAs (on average 0.14 μg). The level of PAs was not associated with the region of Poland, but phytochemical similarity of samples was usually visible at the local scale. Coltsfoot leaves are characterized by a high variability of the content of toxic PAs, much higher than in the case of the main active compounds, especially flavonoids and mucilage. This phytochemical variability is mainly genetically determined (samples came from a garden collection), and it can be increased by environmental factors. Our investigations indicate that Polish natural populations of T. farfara may provide raw material with a low level of toxic PAs.
... ppm. Such a large variation of the amount of PAs seems to be ascribed to a different origin of plant materials, its preparation (raw material or dried samples), extraction methods (with various solvents), and quantification analytical methods (capillary zone electrophoresis, gas, or liquid chromatography) [36,44]. To quantify the PAs in herbal medicines, the selection of appropriate solvent for extraction of PAs and analytical method is considered to be an important work. ...
... μg/g of Senkirkine. Also, Tussilago farfara are reported to contain toxic PAs with various ranges from 0.1 to 368 μg/g [36,44,[46][47][48]. Also, some researchers claim that dry drugs of Tussilago farfara (Farfarae Flos) contain senkirkine from 0.1 to 150 ppm [49,50] and senecionine ranging from 0.1 to 10 ppm [50]. ...
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Pyrrolizidine alkaloids (PAs) that are plant toxin naturally produced for protection against herbivores in some plant families. They are associated with the potential hepatotoxic and carcinogenic diseases serious hepatic disease in humans and animals. As the concern of human health risk posed by exposure to PAs has been gradually increased, precise and reliable analysis is required for monitoring PAs. The present study developed a new and simple pretreatment using 50% MeOH (methanol) for quantification analysis of the PAs contained with high content in the herbal medicines. Another pretreatment method using cation-ion exchange solid-phase extraction (MCX-SPE) was employed for determining most of the PAs that are not contained in the herbal medicines. That is, the proposed LC–MS/MS method coupled with MCX-SPE extraction and 50% MeOH extraction method was developed. And to evaluate the reliability of its application for Farfarae Flos and Lithospermi Radix, a validation study was conducted. In addition, monitory study was performed with ten samples in each herbal medicine. As a result, the proposed method had good linearity with r ² ≥ 0.997. Also, the recoveries indicated to be in the ranges of 70.4–118.0% for the Farfarae Flos, 70.2–119.7% for the Lithospermi Radix. In two herbal medicines, the intra-day precision was revealed to satisfy the reference criteria in most of the PAs. In monitoring results, most of the PAs were not contained in two herbal medicines, whereas a part of PAs revealed to have high concentration in Farfarae Flos and Lithospermi Radix. The proposed method is considered as a simple and reliable method to quantify 28 PAs present in two herbal medicines. Especially, the simple MeOH extraction method seems to be available for quantification analysis of certain PAs in herbal medicines with high content.
... First shots were obtained within 2-4 weeks. The subsequent procedure was applied in accordance with the method described by Wawrosch et al. [16]. Cultures were maintained at standard conditions (temperature of 23±1°C, 16/8 h light/dark photoperiod, illumination of 40-50 mmol m -2 s -1 photosynthetic photon flux intensity provided by cool white fluorescent light) in culture jars (250 ml capacity), 6 explants per each jar. ...
... There is a very limited numbers of reports concerning PAs content in material derived from in vitro cultures. Wawrosch et al. [16] obtained nodal cultures from seeds and found free of alkaloids clone (Wien) that was patented. Micropropagated plants acclimatized and transferred to field conditions produced senkirkine at a level from 0.5 to 46.6 ppm. ...
Article
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Tussilago farfara L. (family Asteraceae) is a valuable medicinal plant that has been used as a cough suppressant and as an antibacterial and anti-inflammatory drug. Mucopolysaccharides, flavonoids, sterols, phenolic acids and pyrrolizidine alkaloids (PAs) are the main active compounds of coltsfoot. Due to hepatotoxic properties of some pyrrolizidine alkaloids, raw materials that contain PAs should be monitored and determined. The aim of present work was to establish nodal cultures of Tussilago farfara and to determine the content of senecionine and senkirkine in plants propagated in in vitro conditions. Eleven clones of coltsfoot derived from Polish natural populations were established. Rhizome buds were used as explants for the initiation of in vitro cultures on MS (Murashige and Skooge) medium. Every six weeks the shots and leaves were collected and dried. The HPLC method was applied for the identification and determination of senecionine and senkirkine. Content of pyrrolizidine alkaloids varied significantly depending on origin (population). An average sum of alkaloids (senecionine and senkirkine) ranged from 1.23 to 10.47 mg/100g d.w. that corresponds to 0.0013–0.011%, respectively.
... Metodą jedno-i dwukierunkowej chromatografii cienkowarstwowej można wstępnie kontrolować, czy w hodowanych organach i zregenerowanych roślinach zachowana została zdolność do biosyntezy wybranych metabolitów wtórnych. Metodami HPLC i GC-MS monitorowana może być także zawartość metabolitów, a w niektórych przypadkach brak toksycznych związków w kulturach [20][21][22]. ...
... [25À29] In many countries the use of such plants in herbal products is prohibited or restricted. [21,30] PAs are toxins, exclusively biosynthesized by plants. They are typical plant secondary metabolites against herbivores. ...
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Tussilago farfara L. (Asteraceae) is widespread in Bulgaria and commonly known as ‘podbel.’ Although T. farfara is a common and widely used herb in folk medicine in the past and today, a study of alkaloid content from Bulgarian populations, which is the aim of this study, has not been done yet. Conventional processing of the dried and powdered plant material (Farfarae folium) including a mild reduction with Zn/HCl of the methanol extracts to convert N-oxides to tertiary bases resulted in the preparation of crude alkaloid mixture (CAM) of pyrrolizidine alkaloids (PAs). The alkaloids were identified by gas chromatography-mass spectrometry analysis of the CAM. A total of four PAs were detected in Farfarae folium herbal substance. Senkirkine and senecionine were found to be major alkaloids, together with integerrimine and seneciphylline, as minor components. The alkaloid content was relatively low (0.0055%) or 55 µg/g. All of the above-mentioned alkaloids were unsaturated at 1,2 position and belonged to the group of the highly hepatotoxic macrocyclic diesters. The content of PAs has to be controlled in processes of T. farfara herbal gathering and herb production, quality control of food, nutrient supplements and other coltsfoot based products. The good botanical identification of T. farfara and morphologically closer PA content species is a prerequisite for quality monitoring and control of plant based products. Microscopic differentiation of the leaves of coltsfoot and butterbur (Petasites spp.) was established and described.
... Most [16]. To achieve this requirement some free of the toxic compounds T. farfara clones were selected and introduced [17]. ...
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Residues obtained after isolating essential oil very often constitute more than 99% of the total raw material. Such residues are poorly exploited although they may represent a potential sustainable source for valuable natural products. This study investigated antioxidant properties and the composition of bioactive compounds (total phenolics, flavonoids, flavonols) present in the deodorized extracts of Tussilago farfara flowers and stems collected in Lithuania and South of France, which were isolated with acetone, methanol or ethanol. Online HPLC/UV/DPPH scavenging assay showed that among 8 identified by HPLC/MS compounds, dicaffeoylquinic acids and quercetin pentoside were the major radical scavengers in the T. farfara extracts.
... Metodą jedno-i dwukierunkowej chromatografii cienkowarstwowej można wstępnie kontrolować, czy w hodowanych organach i zregenerowanych roślinach zachowana została zdolność do biosyntezy wybranych metabolitów wtórnych. Metodami HPLC i GC-MS monitorowana może być także zawartość metabolitów, a w niektórych przypadkach brak toksycznych związków w kulturach202122. Przed założeniem uprawy polowej rośliny leczniczej konieczne jest ustalenie profilu chemicznego mikrosadzonek oraz umieszczenie informacji o chemotypie razem z nazwą botaniczną rośliny. ...
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Plant tissue cultures offer an effective way for a large scale clonal multiplication of superior plants, through pre-existing meristems, with high genetic uniformity and high quality. According to ESCOP, herbal drugs should be obtained from standardized raw material. In the light of new GMP and GAP recommendations it has become necessary to assess shoot cultures and in vitro–derived plants, which may increase the quality of future herbal raw materials. The culture of medicinal plants in controlled in vitro conditions seems to be the best method of certified plantlets production. Especially the “classical” micropropagation techniques are of interest when certain criteria are fulfilled. However, somaclonal variation is observed in plant tissue culture which can lead to changes in content of desired compounds and thus decrease the quality of the plant material. In order to avoid it, each stage of micropropagation should be controlled and plantlets should be assessed in comparison to donor plants. Confirmation of genetic stability is of particular importance in medicinal plants. Quality issues in a tissue culture laboratory also require our special attention. For this reason, the shoot cultures can be evaluated morphologically, cytogenetically, physiologically, biochemically and phytochemically. According to modern standards, the microbiological quality of micropropagated plants is also necessary. It has been showed that in vitro cultures can be evaluated by biological markers and that the first standardization of the plants at the level of in vitro culture is possible. Varied evaluation of micropropagated plants has showed that appropriate protocols of in vitro propagation can be used for a rapid multiplication of superior plant for establishing controlled, ecological cultivations. The controlled cultivation of medicinal plants for the production of herbal medicinal products has gained in importance in recent years due to increased requirements as regards quality and documentation.
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Tussilago farfara L., a perennial species, is a medicinal herb used in traditional medicine, mainly for the treatment of respiratory tract-related pathology. In traditional Chinese medicine, flower buds are preferred; in Europe, the leaves are used; and in some parts of India, the whole plant is utilized. This preferential usage of the plant organs might be based on differences in the chemical composition due to environmental conditions, along with preferred traditional and cultural approaches. In this article, the impact of pedoclimatic growth conditions on the morpho-anatomical development and phytochemical profile of the plant were studied on T. farfara in the vegetative state, collected from two different locations in the Romanian spontaneous flora, revealing significant variations. Furthermore, the antioxidant profile of the specific extracts from the aerial and subterranean plant parts is also in accordance with these discrepancies. The plant anatomy was assessed histologically by optical microscopy, while the analytical chemistry evaluation was based on LC/MS and spectral methods for the evaluation of the antioxidant and enzyme inhibitory activity. To our knowledge, this is the first comparative analysis contextually reporting on the histology, phenolic profile, antioxidant capacity, and geographical location of the vegetative form of T. farfara.
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Ethnopharmacological relevance Farfarae Flos (FF; dried flower buds of Tussilago farfara L.), a widely used traditional Chinese medicine (TCM), is also known as “Kuandonghua” (Chinese: 款冬花). It has a wide range of pharmacological effects and has long been used to treat various respiratory conditions including cough, asthma, and acute or chronic bronchitis. Aims This study reviews the current advances in the research on the botany, ethnopharmacology, phytochemistry, pharmacology, and toxicology of Farfarae Flos. Prospects for future investigation and application of this herb are also discussed. Materials and methods Information on FF was collected from both published materials (such as ancient and modern books, Ph.D. and M. Sc. dissertations, monographs on medicinal plants, and pharmacopoeia) and electronic databases (such as CNKI, SciFinder, WanFang data, PubMed, ScienceDirect, Web of Science, Taylor&Francis, ACS Publications, Wiley, Springer, Europe PMC, EBSCOhost, J-STAGE, and Google Scholar). Results Approximately 175 chemical compounds, including terpenoids, organic acids, flavonoids, alkaloids, chromones, volatile oils, and other compounds, have been isolated from FF. This TCM has been reported to produce pharmacological effects on the respiratory, cardiovascular, and digestive systems, and exert antioxidant, anti-inflammatory, and neuroprotective activities. FF is safe in the traditional dose range, but the potential toxicity due to the emergence of pyrrolidine alkaloids needs to be paid more attention. Conclusions FF is a commonly used TCM with pharmacological activities mainly on the respiratory system. This study suggests that the further expansion of the pharmacological effect of FF and in-depth study of its prescription need to be concerned about. The investigations of the role of more active substances and the pharmacokinetics of the hepatotoxic components of FF will help to maximize the therapeutic potential and promote its popularization and application. Meanwhile, it is important to pay attention to pursue research on the similarities and differences between the leaves and the flower buds to find their respective advantages and make rational use of T. farfara L. resources.
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This review covers a range of pyrrolizidine alkaloids which have been isolated from various natural sources. The synthesis of necines and necic acids is described along with pharmacological and biological studies of pyrrolizidine alkaloids. The literature from July 2000 to June 2001 is reviewed, and 59 references are cited.
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NEWBORN rats have proved strikingly more susceptible than weanlings to the hepatotoxic action of a single dose of the hepatocarcinogen retrorsine, and also to senkirkine and hydroxysenkirkine (cyclic diesters of otonecine). It is usually accepted that substances which induce principally liver tumours require ``activation'' by liver microsomal enzymes before they become ``proximal'' (locally acting) carcinogens. Newborn rats, however, are deficient in enzymes which metabolize drugs, but they are very susceptible to hepatocarcinogens. My work has led me to suggest that the ``activated'' species of pyrrolizidine alkaloids, aflatoxins and certain other carcinogens are the respective epoxides formed on their chemically reactive double bonds. The microsomal enzymes present in older rats probably accelerate the conversion of the epoxides into less active forms.